I encountered a strange prob during one of my assay. Worms do not seem to undergo development on dead bacteria. I heat killed bacteria. I have used temperatures of 70 C for 30 mins and 90 C for 15 mins. The worms seem to be stuck in L2-L3 stage even after a week incubation i wonder why is it so?
I have grown worms using dead OP-50 in liquid media. Might your heat treatment have caused the bacteria to clump, so that they can not be eaten by the larvae? Or might a toxic organism be growing on your killed bacteria that is toxic to the worms?
I have also grown worms using either heat killed or gamma-irradiated bacteria. Worms were always growing fine. Maybe you lyzed your bacteria?
There is no contamination on the plate i have tried to concentrate the bacteria (2X) then heat killed and there was no difference? i grow my worms on agar. At wat temp did u kill the bacteria and for how long?? anyone tried growing worms on agar with dead bacteria??
I found the following reference with a little googling:
They found no difference in life expectancy when worms were grown on dead vs. live OP50 on NGM plates. Another reference mentioned treating the E. coli at 70C for 1 hour to ensure that the bacteria are dead. Maybe this treatment would help?
Is it possible that you are using a strain in which there might be larval arrest, which would have nothing to do with the food issue?
I tried dead OP50 feed before. Providing sufficient food to worms is necessray for their normal development. But heat-killed OP50 couldn’t form nice lawn and proportion of them will never be consumed by worms. That is, worm will has already been of stavation while the lawn still looks sufficient. So, it is highly recommended to transfer worms each two days when you feed them with heat-killed OP50, no matter the lawn consumption.
I have killed my bacteria for an hour at 70 degrees in a waterbath and tried to grow them on LB plates to check if they were dead. They were indeed dead, but I have the same problem as the one started this topic. My worms don’t want to grow on dead OP50 after I synchronized them (N2 and Eat-2), while they same synchronized worms did grow on living OP50. So don’t know what the problem is and putting every 2 days 100 worms on a new plate is a lot work, so is there not a better solution for this?
I know it’s an old-ish thread, but:
this phenomenon has been documented before, at least twice (Jeong, P. Y., Jung, M., Yim, Y. H., Kim, H., Park, M., Hong, E., Lee, W., Kim, Y.H., Kim, K. and Paik, Y. K. (2005). Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature 433,541 -545.) (Anne-Françoise Ruaud and Jean-Louis Bessereau (2006) Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans. Development 133, 2211-2222)
It is, unfortunately, not widely known.
following the line developed in the contributions of LRYoung and Antoineb, it would be useful to know which strain arrests in your assay…and then perhaps it is easier to see how this might tie in with data from the Ruaud & Bessereau paper (which identified a ‘previously uncharacterized L2 diapause’ brought on by feeding the worms dead OP50).
How about killing your e. coli with UV? We use our Stratalinker to UV irradiate our plates, works fine.
thank u for ur references and apologies for the late reply…i use N2 strain…so i guess it ideally should arrest itself in L2…but as the reference provide by antoineb it shows tat there is a L2 dispause stage… but i couldnt find a reason as to why this happens…the references only document the phenomena…it is still intriguing as to why worms selectively do not feed on dead bacteria even when provided in abundance???
@AVSamuelson what is the nm you use when irradiate your plates with UV? I have a 10cm dish, which I have to irradiate. Thanks!
We use a UV Stratalinker 2400, where you can only set the time for UV exposure. I expect for each machine the time that it takes to kill the bacteria will be different. My post-doc did a time course and settled on a 10 minute exposure to UV kill all of the e. coli. Hope this helps and good luck!
May be you can try to kill OP50 with uv, as suggested by AVSamuelson? Alternatively, you may try to use antibiotics, e.g. Kanamycin to kill OP50?
Thanks, but has someone ever killed OP50 in LB medium instead of putting it first onto plates? Because I can’t irradiate plates in which we are putting drugs, because it can affect the drug off course when you use UV. Hopefully you can help me with this issue!